DESCRIPTION (APPLICANT'S ABSTRACT): To ensure precise duplication of the nuclear genome befOre mitotic cell division, a conserved mechanism is employed in all eukaryotes to limit the initiation of DNA synthesis from each replication origin to no more than once in one cell cycle. Defects in the regulation of DNA replication often lead to detrimental consequences such as cell death or uncontrolled cell division, which is manifested in many human cancers. The long-term objective of this proposal is to understand how the initiation of DNA synthesis is regulated. The question will be approached genetically and biochemically in the yeast Saccharomyces cerevisiae, using a novel replication factor, Mcml0, as the probe. Mcm10 is involved in both initiation of DNA replication and the passage of replication forks through unfired origins. Mutations in MCM1O not only reduce the efficiency of origin-specific initiation, but also stall replication forks at unfired origins. We have demonstrated that Mcm1O self-interacts, and that it is a target of regulation by the Cdc28 cyclin-dependent protein kinase. The specific aims of this proposal are: (1) Characterize the self-interaction and phosphorylation ofMcml0. The interaction domain and the phosphorylation site(s) will be mapped. The specific functions of these two post-translational regulatory events will be determined by characterizing mutant alleles defective either in the self-interaction or phosphorylation. (2) Determine specific activities of McmlO at replication origins. ARS1 sequences required for McmlO binding will be determined by in vitro binding assays and in vivo crosslinking. The interaction between McmlO and other replication initiation factors, including Cdc6 and the MCM2-7 complex, will be characterized biochemically. (3) Investigate the roles of McmlO on the replication fork and characterize mcmlO mutants and suppressors. Genetic analysis will be carried out to determine if McmlO also plays a role after the initiation of DNA synthesis. An assay will be developed using mcmlO mutants to monitor the interaction between replication forks and unfired origins. Finally, mutant screens will be carried out to identify new mcmlO mutant alleles and Mcm 10-interacting proteins. These studies will provide a unique perspective to the understanding of origin activity and the interactions between the origin and the replication fork.